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A new method for deconvolution of electrospray ionisation mass spectrometry (ESI-MS) spectra was produced, allowing for the masses of overlapping charge state series to be correctly indentified. The algorithm also determines the abundance of individual molecular species with a much higher accuracy for congested spectra. Several new methods for representing TWIM-MS data were developed. The combination of the deconvolution algorithm with travelling wave ion mobility data creates plots with collision cross section (CCS) axes which can be directly compared with X-ray crystallography structures and computational models. Difference plots have allowed multidimensional analysis of changes in condition, and spectral averaging can produce a single representative spectrum from multiple replicates. Gas-phase unfolding experiments using TWIM-MS are a popular method for probing protein stability in response to conditions such as ligand binding. The algorithms for processing these data are however in their infancy. This thesis describes the first deconvolution algorithm for gas-phase unfolding data, allowing for the accurate interpretation of conformation cross sections and abundances during the unfolding procedure. The methodologies developed were then applied to 1-antitrypsin, a metastable, aggregation prone protein. The protein was bound to a ligand, Ac- TTAI-NH2, which has been shown to block aggregation as a titration and the MS deconvolution method was used to quantify the abundances of each bound state in each mass spectrum. The first use of IM-MS to analyse ex vivo aggregates are shown, and the ion mobility methods created were used to determine the CCS values of the monomeric and dimeric species. The interaction between 1-antitrypsin and Ac-TTAI-NH2 was probed using gas-phase unfolding experiments, determining that the ligand stabilises the protein, with a specific pattern of gas-phase unfolding observed for each state. |
